Maspin-based treatment and prevention of cancer

ABSTRACT

The present invention provides maspin-related compositions and methods of use thereof. In particular, the present invention provides maspin-related compositions, and methods or use thereof, for the promotion of cell adhesion.

CROSS-REFERENCE TO RELATED INFORMATION

The present invention is a divisional of U.S. patent application Ser.No. 13/478,904, filed May 23, 2012, now U.S. Pat. No. 8,791,233, whichclaims the benefit of U.S. Provisional Application Ser. No. 61/489,514,filed May 24, 2011, each of which are incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention provides compositions and methods for thetreatment of prevention of cancer through enhanced cell adhesion. Inparticular the present invention provides maspin-related compositions,and methods or use thereof, for the promotion of cell adhesion.

BACKGROUND

Maspin is a non-inhibitory serine protease inhibitor (serpin) that wasoriginally identified as a type II tumor suppressor protein in mammaryepithelial cells (Zou et al. (1994) Science 263, 526-529; hereinincorporated by reference in its entirety). One major tumor suppressorfunction of maspin is suppression of tumor cell motility, since itinhibits tumor cell migration/invasion in vitro and suppressesmetastasis in mouse models (Zou et al. (1994) Science 263, 526-529;Abraham et al. (2003) J Urol 169, 1157-1161; Seftor et al. (1998) CancerRes 58, 5681-5685; Shi et al. (2001) Cancer Res 61, 6945-6951; Shi etal. (2002) Mol Ther 5, 755-761; Zhang et al. (1997) Mol Med 3, 49-59;Zhang et al. (2000) Oncogene 19, 6053-6058; herein incorporated byreference in their entireties). Several studies show that pericellularmaspin inhibits cell motility by enhancing cell adhesion (Abraham et al.(2003) J Urol 169, 1157-1161; Seftor et al. (1998) Cancer Res 58,5681-5685; Ngamkitidechakul et al. (2001) Invest Ophthalmol Vis Sci 42,3135-3141; Cella et al. (2006) Faseb J 20, 1510-1512; hereinincorporated by reference in their entireties). In addition to its tumorsuppressing functions, maspin is also essential for normal fetaldevelopment since maspin knockout mice are embryonic lethal during theperi-implantation stage partially due to disrupted visceral endodermalcell adhesion (Gao et al. (2004) Development 131, 1479-1489; hereinincorporated by reference in its entirety).

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides a method ofenhancing cell adhesion comprising administering a pro-adhesion proteinor peptide to cells, tissue, or a subject. In some embodiments,enhancing cell adhesion results in reduced cell motility. In someembodiments, the cells, tissue, or subject comprise cancer cells, cellsat risk of becoming cancerous, or cells at risk of metastasis. In someembodiments, the protein or peptide has at least 70% sequence (e.g., 70%. . . 80% . . . 90% . . . 95% . . . 98% . . . 99%) identity with all ora portion of wild-type maspin. In some embodiments, the protein orpeptide has at least 70% sequence (e.g., 70% . . . 80% . . . 90% . . .95% . . . 98% . . . 99%) identity with all or a portion of a mutantmaspin. In some embodiments, the protein or peptide has at least 70%sequence (e.g., 70% . . . 80% . . . 90% . . . 95% . . . 98% . . . 99%)with a portion of maspin that is at least 6 amino acids in length (e.g.,6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 aminoacids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids,15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 aminoacids, 20 amino acids . . . 30 amino acids . . . 40 amino acids, ormore).

In some embodiments, the protein or peptide is capable of physicallyassociating with uPAR, β1 integrin, or both. In some embodiments, theprotein or peptide localizes at the cell surface upon administration tothe cells, tissue, or a subject. In some embodiments, the protein orpeptide co-localizes with the uPA/uPAR complex.

In some embodiments, the protein or peptide comprises a region with 70%sequence identity (e.g., 70% . . . 80% . . . 90% . . . 95% . . . 98% . .. 99%) with SEQ ID NO:1 (TDTKPVQMMNMEA). In some embodiments, theprotein or peptide comprises a region with 70% sequence identity (e.g.,70% . . . 80% . . . 90% . . . 95% . . . 98% . . . 99%) with SEQ ID NO:2(ANAKVKLSIP). In some embodiments, the protein or peptide comprises aregion with 70% sequence identity (e.g., 70% . . . 80% . . . 90% . . .95% . . . 98% . . . 99%) with SEQ ID NO:3 (NPSTMANAKVKLSIPK). In someembodiments, the protein or peptide comprises a region with 70% sequenceidentity (e.g., 70% . . . 80% . . . 90% . . . 95% . . . 98% . . . 99%)with SEQ ID NO:4 (TDTKPVQMMNMEATFCMGNIDSI). In some embodiments, theprotein or peptide comprises a region with 70% sequence identity (e.g.,70% . . . 80% . . . 90% . . . 95% . . . 98% . . . 99%) with SEQ ID NO:5(STANAKVKLSIP). In some embodiments, the protein or peptide comprises aregion with 70% sequence identity (e.g., 70% . . . 80% . . . 90% . . .95% . . . 98% . . . 99%) with SEQ ID NO:6 (TANAEVKLSIPK). In someembodiments, the protein or peptide comprises a region with 70% sequenceidentity (e.g., 70% . . . 80% . . . 90% . . . 95% . . . 98% . . . 99%)with SEQ ID NO:7 (STENAKVKLSIP).

In some embodiments, the present invention provides a compositioncomprising a peptide with at least 70% sequence identity with SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6and/or SEQ ID NO:7, wherein the peptide enhances cell adhesion whenadministered to cells, tissue, or a subject.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising: (a) a protein or peptide with at least 70%sequence identity with all or a portion of wild-type maspin, and (b) aphysiologically suitable buffer. In some embodiments, the protein orpeptide with at least 70% sequence identity with all or a portion ofwild-type maspin enhances cell adhesion when administered to cells,tissue, or a subject. In some embodiments, all or a portion of theprotein or peptide comprises at least 70% sequence identity with one ormore of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, and/or SEQ ID NO:7.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising: (a) a peptide with at least 70% sequenceidentity with SEQ ID NO:1; (b) a peptide with at least 70% sequenceidentity with SEQ ID NO:2; and (c) a physiologically suitable buffer. Insome embodiments, the pharmaceutical composition enhances cell adhesionwhen administered to cells, tissue, or a subject.

In some embodiments, the present invention provides a pharmaceuticalcomposition comprising: (a) one or more of: (i) a peptide with at least70% sequence identity with SEQ ID NO:1, (ii) a peptide with at least 70%sequence identity with SEQ ID NO:2, (iii) a peptide with at least 70%sequence identity with SEQ ID NO:3, (iv) a peptide with at least 70%sequence identity with SEQ ID NO:4, (v) a peptide with at least 70%sequence identity with SEQ ID NO:5, (vi) a peptide with at least 70%sequence identity with SEQ ID NO:6; (vii) a peptide with at least 70%sequence identity with SEQ ID NO:7; and (b) a physiologically suitablebuffer. In some embodiments, the pharmaceutical composition enhancescell adhesion when administered to cells, tissue, or a subject.

In some embodiments, the present invention provides a method of treatingor preventing cancer or metastasis comprising administering to a cell,tissue, or subject a composition comprising a protein or peptidecomprising at least 70% sequence identity with a or a portion of one ormore of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, or all or a portion of wild-type maspin,wherein administering the protein or peptide enhances cell adhesionand/or inhibits cell motility.

The present invention is not limited to treatment and/or prevention ofcancer. The peptides provided herein also find use in treating otherdiseases and disorders. In certain embodiments, the present inventionprovides methods for treating bone formation disorders (e.g., byadministration of peptides provided herein). In some embodiments, themethods comprise administering to a subject suffering from a boneformation disorder a composition comprising a protein or peptidecomprising at least 70% sequence identity with a or a portion of one ormore of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, or all or a portion of wild-type maspin.The methods are not limited to a particular type or severity of a boneformation disorder. An example of a bone formation disorder includes,but is not limited to, osteoporosis. The methods are not limited totreating a certain type of subject. In some embodiments, the subject isa rodent (e.g., mouse), while in other embodiments; the subject is ahuman being. In some embodiments, the composition is coadministeredwith, for example, an anti-osteoporosis agent (e.g., a hormonereplacement therapy agent, a bisphosphonate (e.g., alendronate (e.g.,FOSAMAX)), vitamin D, an androgen, a parathyroid hormone, a selectiveestrogen-receptor modulators, and a calcitonin-salmon). In certainembodiments, osteoblast cell proliferation is promoted by administeringto a sample (or subject) comprising osteoblast cells a compositioncomprising a polypeptide or peptide comprising at least 70% sequenceidentity with a or a portion of one or more of: SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,or all or a portion of wild-type maspin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows images of Western blots demonstrating maspin release fromMCF10A cells. Human corneal epithelial cells (hCEC), used as a control,or low and high passage MCF10A cells were maintained in definedkeratinocyte serum free medium. Conditioned medium was collected,centrifuged to remove cell debris, and concentrated. Cells (2×106) weresolubilized with RIPA buffer containing protease inhibitors. Proteinlevels were measured by Pierce Coomassie Reagent and protein (20 μg) wasrun on 10% SDS-PAGE. Proteins in 50 ml of the 24× conditioned mediumwere separated on SDS-PAGE and visualized by Western blots using mouseanti-maspin (BD Biosciences) or rabbit anti-GAPDH (Chemicon).

FIG. 2 shows graphs demonstrating that maspin-derived peptides andmutation of maspin (E201, K268, and/or K270) inhibits MCF10A celladhesion to self-deposited matrix. (A) MCF10A cells were harvested withenzyme-free dissociation buffer, and pre-incubated with the indicatedpeptides. Cells (2.0×104 cells) were seeded and adhesion was measuredafter 30 min using colorimetric reaction. Peptides 181-202, 190-211, and260-275 inhibited cell adhesion whereas no significant effect wasdetected with other peptides. (B) Software Molsoft ICM-pro version 3.48was used for 3D structure analysis of maspin. The amino acids E201(blue), K268, K270 (green) are exposed on the surface of maspin. Thecontrol peptide is marked in red. (C) MCF10A cells (2.0×104 cells) wereincubated with GST, GST-maspin, or GSTmaspin mutants on endogenousmatrix. Maspin mutants, E201K, double mutant (K268E, K270E), and triplemutant (E201K, K268E, K270E) inhibited cell adhesion.

FIG. 3 shows graphs demonstrating that uPAR and uPA/uPAR complex arerequired for maspin localization to MEF cell surface. (A) Wildtype (WT,black circles) and uPAR−/− (white circles) MEFs were incubated with¹²⁵I-GST-maspin in DMEM containing 5% BSA or at 4° C. for 90 min. Cellsurface binding was determined by measuring gamma radiation. (B)Pretreatment of ¹²⁵I-GST-maspin with two different maspin antibodiesshows that the RCL region is necessary for maspin binding to WT MEF cellsurface. The ABS4A antibody (black bars) blocks the RCL region while theS-20 antibody (white bars, Santa Cruz Biotechnology) blocks theN-terminal region. (C) Inhibition of maspin binding to WT MEF cellsurface by the antimouse uPAR antibody (R&D), which blocks uPA bindingto uPAR. WT MEF cells were treated with the uPAR antibody either withoutuPA stripping or with uPA stripping and addition of exogenous uPA.Results are reported as GST-maspin binding as a percentage of control(IgG) antibody treatment.

FIG. 4 shows graphs demonstrating that the uPA and uPA/uPAR complex arenecessary for maspin-mediated cell adhesion. (A) Stripping endogenousuPA from receptor on cell surface decreases maspin guided cell adhesionto the self-deposited matrix. The GST-maspin-mediated increase in MCF10Acell adhesion was rescued by treatment with exogenous uPA (800 nM).Endogenous uPA was first stripped from MCF10A cell surface, then cellswere pretreated with anti-uPAR or rabbit IgG (as a control) at theindicated concentrations for 10 min. Exogenous uPA (800 nM) plusGST-maspin or GST (as a control) were added for another 20 min. MCF10Acells (2.0×104) were plated on endogenous matrix and cell adhesion wasquantified. (C) MCF10A cells (2.0×104) were pretreated with anti-uPAR orrabbit IgG (as a control) at the indicated concentrations for 10 min.GST-maspin or GST (as a control) were added for another 20 min wereplated on endogenous matrix and cell adhesion was quantified.

FIG. 5 shows co-immunoprecipitation of maspin with uPAR and 131integrin. Protein extracts (500 μg) from MCF10A cells overexpressinguPAR were immunoprecipitated with antibodies to uPAR (AmericanDiagnostics), maspin (Pharmingen), and β1 integrin (Chemicon). The Ki67antibody (Chemicon) was used as a negative control. Elutedimmunoprecipitates were separated by SDS-PAGE and analyzed by Westernimmunoblots.

FIG. 6 shows the 3D structure of mapsin. Inset (right panel) indicatesthat three key amino acids E201, K270, K268 are critical for maspinaction.

FIG. 7 shows the effect of maspin peptides X and Y on HUVEC tubeformation. HUVECs were plated on MatriGel for tube formation assay.Various peptides were tested for the potency on tube angiogenesis.Peptide X and Y were shown to be highly potent compared to the G-Helixpeptide or GST-maspin.

DEFINITIONS

As used herein, the term “subject” refers to any human or animal (e.g.,non-human primate, rodent, feline, canine, bovine, porcine, equine,etc.). For methods of treatment, a subject may be any human or animalsubject having a neoplasia, such as cancer or precancer. For methods ofprevention, the subject may be any human or animal subject who is atrisk of metastasis or developing a cancer. The subject may be at riskdue to exposure to carcinogenic agents; being genetically predisposed todisorders characterized by unwanted, rapid cell proliferation; beingpredisposed (e.g., genetically or otherwise) to increased cell motility;etc. The term “patient” is used herein to refer to a human subjectreceiving treatment for a disease, disorder, and or condition, or beingadministered compositions of the present invention.

As used herein, the term “effective amount” refers to the amount of acomposition (e.g., maspin-derived or maspin-related protein or peptide)sufficient to effect beneficial or desired results. An effective amountcan be administered in one or more administrations, applications ordosages and is not intended to be limited to a particular formulation oradministration route.

As used herein, the term “administration” refers to the act of giving adrug, prodrug, or other agent, or therapeutic treatment (e.g.,compositions of the present invention) to a subject (e.g., a subject orin vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplaryroutes of administration to the human body can be through the eyes(ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs(inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g.,intravenously, subcutaneously, intratumorally, intraperitoneally, etc.)and the like.

As used herein, the terms “co-administration” and “co-administer” referto the administration of at least two agent(s) (e.g., a combinationmaspin-derived or maspin-related proteins or peptides, a combination ofoligonucleotides coding for a maspin-derived or maspin-related proteinsor peptides, a maspin-related therapy and one or more other agents,etc.) or therapies to a subject. In some embodiments, theco-administration of two or more agents or therapies is concurrent. Inother embodiments, a first agent/therapy is administered prior to asecond agent/therapy. Those of skill in the art understand that theformulations and/or routes of administration of the various agents ortherapies used may vary. The appropriate dosage for co-administrationcan be readily determined by one skilled in the art. In someembodiments, when agents or therapies are co-administered, therespective agents or therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents or therapies lowers the requisite dosage of a potentially harmful(e.g., toxic) agent(s).

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent (e.g., maspin-derived or maspin-relatedprotein or peptide) with a carrier, inert or active, making thecomposition especially suitable for diagnostic or therapeutic use invitro, in vivo or ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, emulsions (e.g., such asan oil/water or water/oil emulsions), and various types of wettingagents, any and all solvents, dispersion media, coatings, sodium laurylsulfate, isotonic and absorption delaying agents, disintigrants (e.g.,potato starch or sodium starch glycolate), and the like. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. (1975), incorporated herein by reference).

As used herein, the term “pharmaceutically acceptable salt” refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound of the present invention that is physiologically tolerated inthe target subject (e.g., a mammalian subject, and/or in vivo or exvivo, cells, tissues, or organs). “Salts” of the compounds of thepresent invention may be derived from inorganic or organic acids andbases. Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for thetreatment of prevention of cancer through enhanced cell adhesion. Inparticular the present invention provides maspin-related compositions,and methods or use thereof, for the promotion of cell adhesion.

Tumor metastasis involves recognition, degradation, and migrationthrough the surrounding extracellular matrix (ECM), a processintrinsically dependent on cell-ECM adhesion. Many different proteins(and/or protein complexes) are involved in promoting or inhibiting tumormetastasis. Maspin is a tumor suppressing protein that is abundantlyproduced in normal mammary luminal epithelial and myoepithelial cells(Zou et al. (1994) Science 263, 526-529; herein incorporated byreference in its entirety). Upon tumor progression, maspin expression issignificantly reduced or lost in breast and prostate carcinoma celllines and tissues (Zou et al. (1994) Science 263, 526-529; Seftor et al.(1998) Cancer Res 58, 5681-5685; Zhang et al. (1997) Mol Med 3, 49-59;Sheng et al. (1996) Proc Natl Acad Sci USA 93, 11669-11674; hereinincorporated by reference in their entireties). One of the major tumorsuppressing functions of maspin is its ability to inhibit tumor cellmotility and invasiveness, which is partially mediated by enhancing celladhesion. The region between amino acids residues 139-225 in maspinfacilitates increased mammary luminal epithelial (MCF10A) cell adhesion(Cella et al. (2006) Faseb J 20, 1510-1512; herein incorporated byreference in its entirety). Experiments conducted during development ofembodiments of the present invention demonstrate (1) a unique regionproximal to the reactive center loop (RCL) of maspin that mediates itsadhesive function, and (2) identified a novel maspin/uPA/uPAR/β1integrin complex, which facilitates maspin-mediated cell adhesion.Experiments conducted during development of embodiments of the presentinvention implicate maspin as an integrator of the urokinase activationsystem and integrin receptors by which cell adhesion and migration areregulated. Two different regions were identified proximal to the RCL ofmaspin that are responsible for maspin-mediated MCF10A cell adhesion.Enhanced adhesion was found to be dependent on the presence of both uPAand uPAR and is present in a complex with uPA/uPAR/β1 integrin on thecell surface. Together, experiments conducted during development of thepresent invention indicate that maspin coordinates both uPA/uPAR and β1integrin receptors to regulate both mammary epithelial cell-ECM adhesionand migration.

Exogenous maspin treatment elicits similar anti-migratory effects seenin breast carcinoma cells overexpressing maspin cDNA (Seftor et al.(1998) Cancer Res 58, 5681-5685; Sheng et al. (1996) Proc Natl Acad SciUSA 93, 11669-11674; herein incorporated by reference in theirentireties). These findings indicate that both endogenous and exogenousmaspin act similarly at an extracellular site or are transported into anintracellular site of action. Studies suggest that maspin is secreted orpresent/associated at the cell surface (Seftor et al. (1998) Cancer Res58, 5681-5685; Ngamkitidechakul et al. (2001) Invest Ophthalmol Vis Sci42, 3135-3141; Cella et al. (2006) Faseb J 20, 1510-1512; Bass et al.(2009) J Biol Chem 284, 27712-27720; Pemberton et al. (1997) J HistochemCytochem 45, 1697-1706; Khalkhali-Ellis & Hendrix (2007) Cancer Res 67,3535-3539; Sheng et al. (1996) Proc Natl Acad Sci USA 93, 11669-11674;Law et al. (2005) J Biol Chem 280, 22356-22364; herein incorporated byreference in their entireties). However, recent data refutes theseclaims stating that maspin expression is absent from the surface ofMCF10A cells and not secreted through the classical secretion pathway(Teoh et al. (2010) J Biol Chem 285, 10862-10869; herein incorporated byreference in its entirety). Confocal microscopy has been used todemonstrate that maspin is present on MCF10A cell surface and mediatesadhesion (Cella et al. (2006) Faseb J 20, 1510-1512). Experiments wereconducted during development of embodiments to clarify prior conflictingresults on the presence of maspin at the cell surface. Since expressionof maspin in human coronary stromal cells is reduced to undetectablelevels in upon culturing (Ngamkitidechakul et al. (2001) InvestOphthalmol Vis Sci 42, 3135-3141; herein incorporated by reference inits entirety), it was investigated whether maspin secretion is lost as aresult of culturing conditions. The detection of maspin protein, byWestern immunoblot, is reduced in media obtained from high passageMCF10A cells when compared to low passage cells. Additionally, nosignificant difference was detected between cellular maspin expressionsin the high versus low passage MCF10A cells. These data indicate thatextracellular maspin detection is lost depending on culturingconditions; thereby providing an explanation for previous conflictingresults.

Experiments were conducted during development of embodiments of thepresent invention to determine the region involved in maspin-mediatedcell adhesion. Several peptides were generated from the 139-225 regionof maspin. The crystal structure of maspin reveals several structuralmotifs within this region including the third strand of the A β-sheet(s3A), third strand of the C β-sheet (s3C), and first strand of the Bβ-sheet (s1B) (Law et al. (2005) J Biol Chem 280, 22356-22364;Al-Ayyoubi et al. (2004) J Biol Chem 279, 55540-55544; hereinincorporated by reference in its entirety). Due to the presence ofsurface-exposed amino acid side chains, s3C and s1B (amino acids180-210) were tested for adhesion effects. Experiments indicated thats1B (peptides 181-202 and 190-211) not s3C (peptide 169-189) wasimportant in mediating cell adhesion. Moreover, a point mutation (E201K)within this region significantly reduced MCF10A cell adhesion.Experiments indicate that the amino acids overlapping these peptides,amino acids 190-202 (TDTKPVQMMNMEA; SEQ ID NO:1) in maspin are integralfor maspin-mediated cell adhesion. These amino acids are highlyconserved between human, mouse, rat, and chicken species (84% sequenceidentity and 100% similarity), indicating their importance throughoutevolution (Law et al. (2005) J Biol Chem 280, 22356-22364; incorporatedherein by reference in its entirety).

Maspin inhibits prostate carcinoma cell migration and invasion due, atleast in part, to the strengthening of mature focal adhesion contacts(Yin et al. (2006) Cancer Res 66, 4173-4181; herein incorporated byreference in its entirety). In addition, experiments conducteddemonstrate that maspin binds uPA (and pro-uPA) and co-localizes withuPAR on the cell surface (Yin et al. (2006) Cancer Res 66, 4173-4181;McGowen et al. (2000) Cancer Res 60, 4771-4778; Biliran & Sheng. (2001)Cancer Res 61, 8676-8682; herein incorporated by reference in theirentireties). Maspin binding does not directly inhibit uPA proteolyticactivity but rather binds to uPA (and pro-uPA) via a region of maspin inclose proximity to the RCL (Al-Ayyoubi et al. (2007) J Biol Chem 282,19502-19509; Bass et al. (2002) J Biol Chem 277, 46845-46848; hereinincorporated by reference in their entireties). Experiments conductedduring development of the present invention demonstrated that the RCL isin close proximity to the s1B (191-211) region of maspin. Anotherregion, s2C (SEQ ID NO:2; residues 265-274; ANAKVKLSIP), is alsoproximal to both the RCL and s1B. Experiments conducted duringdevelopment of embodiments of the present invention demonstrated thatthe s1B region is important in mediating cell adhesion. Moreover, the260-275 peptide (SEQ ID NO:3; NPSTMANAKVKLSIPK) and double mutant GSTmaspin (K268E, K270E) reduced MCF10A cell adhesion compared to wild-typeGST-maspin. Experiments using the triple mutant maspin (E201K, K268E,K270E) verified that both regions are important for cell adhesion. Thesefindings indicate that the s1B and s2C regions of maspin facilitate celladhesion.

Experiments were conducted during development of embodiments of thepresent invention to identify dependence of maspin-mediated celladhesion on the plasminogen activation pathway. Removal of endogenousuPA from the surface of MCF10A cells abrogated maspin-mediated adhesion.However, administration of exogenous uPA restored maspin-mediated celladhesion in a concentration-dependent manner. These results indicatethat that exogenous (or cell surface localized) uPA expression isnecessary for the proadhesion function of maspin.

Localization of uPA to cell-cell contacts and focal adhesions ismediated by its receptor, uPAR. The uPA/uPAR interaction is very tightwith binding affinities in the low nanomolar range. Extracellular maspinexpression is localized to the cell surface (Abraham et al. (2003) JUrol 169, 1157-1161; Yin et al. (2006) Cancer Res 66, 4173-4181; Biliran& Sheng. (2001) Cancer Res 61, 8676-8682; Pemberton et al. (1997) JHistochem Cytochem 45, 1697-1706; herein incorporated by reference intheir entireties). Experiments conducted during development ofembodiments of the present invention to evaluate the binding ofexogenous maspin to wild type (WT) or uPAR−/− mouse embryo fibroblast(MEFs) found maspin localized on the surface of WT MEFs, but did notdetect maspin expression on the surface of uPAR−/− MEFs. These resultsindicate that maspin binds to uPA (and potentially pro-uPA) and islocalized to the cell surface by maspin/uPA binding to uPAR. Experimentswere conducted to confirm the importance of the uPA/uPAR complex infacilitating maspin-mediated cell adhesion by: (1) stripping uPA fromthe surface of MCF10A cells, (2) disrupting the uPA/uPAR binding with anantibody, and (3) testing MCF10A cell adhesion after uPA and GSTmaspinwere added. These experiments demonstrated that if uPA is blocked frombinding to uPAR, the increased MCF10A cell adhesion mediated by maspinis significantly reduced. Alternatively, if uPA is not stripped from thecell surface and the uPAR antibody is added, no effect in adhesion wasdetected. These results indicate that maspin localization to the cellsurface and function on cell adhesion is based on the uPA/uPAR complex.As such, altered cell surface expression of uPA or uPAR will result inaltered maspin localization and cell adhesion. For instance, cells undersiege from increased phospholipase and protease activity liberate uPARfrom its GPI membrane tether (Sidenius et al. (2000) FEBS Lett 475,52-56; Wilhelm et al. (1999) J Cell Physiol 180, 225-235; hereinincorporated by reference in their entireties).

Experiments conducted during development of the present inventiondemonstrate that maspin produces a novel coordination of the plasminogenactivation system and integrin receptors, which is necessary tofacilitate its anti-migratory ability. The s1B and s2C regions (aminoacid residues 190-202 and 260-275, respectively) of maspin areresponsible for its effects on cell adhesion and the necessity of theuPA/uPAR complex for this effect. The ability of maspin to integrateinto this signaling complex is retained even in tumor cells, asrecombinant maspin still facilitates anti-migratory signaling even afterits expression is lost (Zou et al. (1994) Science 263, 526-529; Seftoret al. (1998) Cancer Res 58, 5681-5685; Sheng et al. (1996) Proc NatlAcad Sci USA 93, 11669-11674; Sheng et al. (1996) Proc Natl Acad Sci USA93, 11669-11674; Zhang et al. (1999) Dev Biol 215, 278-287; hereinincorporated by reference in their entireties).

In some embodiments, the present invention provides compositions, kits,systems, and/or methods to treat or prevent cancer, metastatasis, orunusual cell motility. In some embodiments, the present inventionenhances cell adhesion and/or prevents cell motility. In someembodiments, cell adhesion is enhanced in cells undergoing metastasis orat risk of entering metastasis. In some embodiments, cell adhesion isenhanced in neoplastic cells (e.g., cancerous, pre-cancerous). In someembodiments, compositions and methods are utilized in the treatmentand/or prevention of: bladder cancer, lung cancer, breast cancer,melanoma, colon and rectal cancer, non-Hodgkin lymphoma, endometrialcancer, pancreatic cancer, kidney (renal cell) cancer, prostate cancer,leukemia, thyroid cancer, and/or metastasis thereof. In someembodiments, the present invention enhances cell adhesion and/orinhibits cell motility in non-neoplastic or non-cancerous cells.

In some embodiments, the present invention provides a pharmaceuticals,small molecules, peptides, proteins, polypeptides, nucleic acids,molecular complexes, etc. for the treatment or prevention of cancer(e.g., metastasis), enhancement of cell adhesion, and/or inhibition ofcell motility. In some embodiments, the present invention providesadministration of a maspin-based peptide (e.g., polypeptide comprising:full length maspin, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, etc.) to inhibit cell motilityand/or enhance cell adhesion. In some embodiments, a polypeptide of thepresent invention can be prepared by methods known to those of ordinaryskill in the art. For example, the claimed polypeptide can besynthesized using solid phase polypeptide synthesis techniques (e.g.Fmoc). Alternatively, the polypeptide can be synthesized usingrecombinant DNA technology (e.g., using bacterial or eukaryoticexpression systems). Accordingly, to facilitate such methods, thepresent invention provides genetic vectors (e.g., plasmids) comprising asequence encoding the inventive polypeptide, as well as host cellscomprising such vectors. Furthermore, the invention provides thepolypeptide produced via recombinant methods.

In some embodiments, the present invention provides administration ofmaspin-based (e.g., maspin-related, maspin-derived, etc.) compositions(e.g. maspin, maspin-based peptides, mimetics of maspin, nucleic acidsencoding maspin-based peptides, etc.). In some embodiments, the presentinvention provides administration of polypeptides which inhibit cellmotility (e.g. maspin, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, combinations thereof,derivatives thereof, etc.). In some embodiments, the present inventionprovides administration of nucleic acids which encode polypeptidesmotility (e.g. maspin, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, combinations thereof,derivatives thereof, etc.) which inhibit cell motility and/or enhancecell adhesion. In some embodiments, a polypeptide comprising orconsisting of SEQ ID NO:1 is administered. In some embodiments, apolypeptide comprising or consisting of SEQ ID NO:2 is administered. Insome embodiments, a polypeptide comprising or consisting of SEQ ID NO:3is administered. In some embodiments, a polypeptide comprising orconsisting of SEQ ID NO:4 is administered. In some embodiments, apolypeptide comprising or consisting of SEQ ID NO:5 is administered. Insome embodiments, a polypeptide comprising or consisting of SEQ ID NO:6is administered. In some embodiments, a polypeptide comprising a portionwith at least 50% homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, and/or SEQ ID NO:7 is administered(e.g. at least 60% homology, at least 70% homology, at least 80%homology, at least 90% homology, at least 95% homology, at least 99%homology, etc.). In some embodiments, administering a maspin-basedpeptide, nucleic acid, or a drug-like small molecule to a subject orcell inhibits pathways related to cell-motility, inhibits cell motility,and/or protects against undesired cell motility.

In some embodiments, polypeptides of the present invention are isolatedand/or purified (or substantially isolated and/or substantiallypurified). Accordingly, the invention provides polypeptide insubstantially isolated form. In some embodiments, polypeptides areisolated from other polypeptides as a result of solid phase proteinsynthesis, for example. Alternatively, polypeptides can be substantiallyisolated from other proteins after cell lysis from recombinantproduction. Standard methods of protein purification (e.g., HPLC) can beemployed to substantially purify polypeptides. In some embodiments, thepresent invention provides a preparation of polypeptides in a number offormulations, depending on the desired use. For example, where thepolypeptide is substantially isolated (or even nearly completelyisolated from other proteins), it can be formulated in a suitable mediumsolution for storage (e.g., under refrigerated conditions or underfrozen conditions). Such preparations may contain protective agents,such as buffers, preservatives, cryprotectants (e.g., sugars such astrehalose), etc. The form of such preparations can be solutions, gels,etc., and the inventive polypeptide can, in some embodiments, beprepared in lyophilized form. Moreover, such preparations can includeother desired agents, such as small molecules or even other polypeptidesand proteins, if desired. Indeed, the invention provides such apreparation comprising a mixture of different embodiments of theinventive polypeptide (e.g., a plurality of polypeptide species asdescribed herein).

In some embodiments, the present invention also provides apharmaceutical composition comprising of one or more polypeptides(full-length maspin or portion thereof, polypeptides comprising SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and/or SEQ IDNO:6, mixtures thereof, derivatives thereof, mutants thereof, etc.) anda pharmaceutically acceptable carrier. Any carrier which can supply apolypeptide without destroying the vector within the carrier is asuitable carrier, and such carriers are well known in the art. Thecomposition can be formulated for parenteral, oral, or topicaladministration. For example, a parenteral formulation could consist of aprompt or sustained release liquid preparation, dry powder, emulsion,suspension, or any other standard formulation. An oral formulation ofthe pharmaceutical composition could be, for example, a liquid solution,such as an effective amount of the composition dissolved in diluents(e.g., water, saline, juice, etc.), suspensions in an appropriateliquid, or suitable emulsions. An oral formulation could also bedelivered in tablet form, and could include excipients, colorants,diluents, buffering agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible excipients. A topicalformulation could include compounds to enhance absorption or penetrationof the active ingredient through the skin or other affected areas, suchas dimethylsulfoxide and related analogs. The pharmaceutical compositioncould also be delivered topically using a transdermal device, such as apatch, which could include the composition in a suitable solvent systemwith an adhesive system, such as an acrylic emulsion, and a polyesterpatch. Compositions could be delivered via eye drops or other topicaleye delivery method. Compositions may be delivered intraocularly,anywhere in the eye including, for example, the vitreous cavity, theanterior chamber, etc. Compositions may be delivered intravitrealy as iscommonly done with intravitreal injections of Lucentis (ranabizumab),Avastin (bevazizumab), triamcinolone acetonide, antibiotics, etc.Compositions may be administered using encapsulated cell technology(e.g. by Neurotech) in which genetically modified cells are engineeredto produce and secrete compositions of the present invention (e.g.maspin-based proteins or peptides).

In some embodiments, the methods of the present invention are employedin vivo. In some embodiments, polypeptides are delivered to a human oranimal subject in an amount and at a location sufficient to inhibit orattenuate cell motility or enhance cell adhesion within a population ofcells (e.g., within desired tissue, within the patient, etc.).Polypeptide can be formulated into a suitable pharmaceutical composition(e.g., as described above or as otherwise known to those of ordinaryskill in the art) for delivery into the subject. The delivery can belocal (e.g., by injection or implantation within the desired tissue tobe treated) or systemic (e.g., by intravenous or parenteral injection).

In some embodiments, the present invention provides a method fortreating patients suffering from (or at risk of) neoplasia and in needof treatment (or preventative therapy). In some embodiments, apharmaceutical composition comprising at least one polypeptide of thepresent invention is delivered to such a patient in an amount and at alocation sufficient to treat the condition. In some embodiments,polypeptides of the present invention (or pharmaceutical compositioncomprising such) can be delivered to the patient systemically orlocally, and it will be within the ordinary skill of the medicalprofessional treating such patient to ascertain the most appropriatedelivery route, time course, and dosage for treatment. It will beappreciated that application of the inventive method of treating apatient most preferably substantially alleviates or even eliminates suchsymptoms; however, as with many medical treatments, application of theinventive method is deemed successful if, during, following, orotherwise as a result of the inventive method, the symptoms of thedisease or disorder in the patient subside to an ascertainable degree.

A pharmaceutical compound may be administered in the form of acomposition which is formulated with a pharmaceutically acceptablecarrier and optional excipients, adjuvants, etc. in accordance with goodpharmaceutical practice. The maspin-based (e.g., comprising SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and/orSEQ ID NO:7, mixtures thereof, derivatives thereof, mutants thereof,etc.) pharmaceutical composition may be in the form of a solid,semi-solid or liquid dosage form: such as powder, solution, elixir,syrup, suspension, cream, drops, paste and spray. As those skilled inthe art would recognize, depending on the chosen route of administration(e.g. pill, injection, etc.), the composition form is determined. Ingeneral, it is preferred to use a unit dosage form of the inventiveinhibitor in order to achieve an easy and accurate administration of theactive pharmaceutical compound. In general, the therapeuticallyeffective pharmaceutical compound is present in such a dosage form at aconcentration level ranging from about 0.5% to about 99% by weight ofthe total composition: i.e., in an amount sufficient to provide thedesired unit dose. In some embodiments, the pharmaceutical compositionmay be administered in single or multiple doses. The particular route ofadministration and the dosage regimen will be determined by one of skillin keeping with the condition of the individual to be treated and saidindividual's response to the treatment. In some embodiments, amaspin-based pharmaceutical composition in a unit dosage form foradministration to a subject, comprising a pharmaceutical compound (e.g.,comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 and/or SEQ ID NO:7, mixtures thereof, derivativesthereof, mutants thereof, etc.) and one or more nontoxicpharmaceutically acceptable carriers, adjuvants or vehicles. The amountof the active ingredient that may be combined with such materials toproduce a single dosage form will vary depending upon various factors,as indicated above. A variety of materials can be used as carriers,adjuvants and vehicles in the composition of the invention, as availablein the pharmaceutical art. Injectable preparations, such as oleaginoussolutions, suspensions or emulsions, may be formulated as known in theart, using suitable dispersing or wetting agents and suspending agents,as needed. The sterile injectable preparation may employ a nontoxicparenterally acceptable diluent or solvent such as sterile nonpyrogenicwater or 1,3-butanediol. Among the other acceptable vehicles andsolvents that may be employed are 5% dextrose injection, Ringer'sinjection and isotonic sodium chloride injection (as described in theUSP/NF). In addition, sterile, fixed oils may be conventionally employedas solvents or suspending media. For this purpose, any bland fixed oilmay be used, including synthetic mono-, di- or triglycerides. Fattyacids such as oleic acid can also be used in the preparation ofinjectable compositions.

In some embodiments, maspin-based compositions of the present inventionare provided as part of a kit. In some embodiments, a kit of the presentinvention comprises one or more maspin-based compositions and/ormaspin-based pharmaceutical compositions. In some embodiments, a kitcomprises a maspin-based composition configured for co-administrationwith one or more additional compositions (e.g. pharmaceuticalcompositions). In some embodiments, one or more maspin-basedcompositions are co-administered with one or more other agents foreffective enhancement of cell adhesion, inhibition of cell motility,and/or treatment or prevention of cancer or metastasis.

In some embodiments, maspin-related compositions are provided for thepromotion of cell adhesion and/or inhibition of cell migration. However,the compositions and methods described herein are not limited by theirparticular application or field of use. In some embodiments,compositions and methods find use in the treatment and/or prevention ofcancer, metastasis, or other neoplastic disorders of conditions. In someembodiments, compositions and methods find use in the treatment and/orprevention disorders associated with bone formation (e.g.,osteoporosis), as is described for purified maspin in US Pat. App.20090263363; herein incorporated by reference in its entirety. Variousmodification, recombination, and variation of the described features andembodiments will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although specificembodiments have been described, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes andembodiments that are obvious to those skilled in the relevant fields areintended to be within the scope of the following claims.

EXPERIMENTAL Example 1 Materials and Methods

Antibodies and Reagents—

Rabbit antihuman uPAR (American Diagnostics) for immunoprecipitation(IP) and functional blocking experiments. Mouse monoclonal anti-maspin(Pharmingen) and rabbit polyclonal anti-131 integrin (Chemicon) was usedfor both IP and immunoblot probing. An affinity-purified rabbitpolyclonal antibody raised against maspin reactive center loop (RCL)peptide (AbS4A) was used from previous studies (Zou et al. (1994)Science 263, 526-529). Both the horseradish peroxidase-conjugatedsecondary antibodies and maspin peptides were obtained fromSigma-Aldrich. Human uPA was purchased from Chemicon.

Cell Culture—

MCF10A, immortalized human mammary luminal epithelial cells, (CRL-10317;American Type Culture Collection) were cultured in Dulbecco's modifiedEagle medium (DMEM)/F12 (Invitrogen) containing 5% donor horse serum, 20μg/ml epidermal growth factor (EGF), 100 μg/ml cholera toxin, 10 μg/mlinsulin, 500 μg/ml hydrocortisone, 50 Um′ penicillin, and 50 μg/mlstreptomycin at 37° C. and 5% CO2. All growth factors and hormones werepurchased from Sigma. For maspin secretion studies, MCF10A cells eitherdirectly ordered from ATCC (low passage) or carried in the lab for twoyears (high passage) were maintained in defined keratinocyte serum freemedium (Invitrogen) supplemented with ciprofloxacin and MITO+(BDBiosciences). Cells were passaged weekly and fed three times per week.Conditioned medium was collected on the day of passage, centrifuged toremove cell debris and concentrated 24× using a 30 kDa spin filter(Millipore). Harvested protein was then used for Western blot analysis,as described later. Control (uPAR+/+) and uPAR-deficient (uPAR−/−)murine embryonic fibroblasts (MEFs) were isolated from embryos aspreviously described (Ma et al. (2002) J Cell Biol 159, 1061-1070;herein incorporated by reference in its entirety).

Construction, Expression, and Purification of GST-Maspin and Mutants—

Glutathione Stransferase (GST)-tagged maspin (GST-maspin) was producedas previously described (Zhang et al. (2000) Nat Med 6, 196-199; hereinincorporated by reference in its entirety). Point mutants wereconstructed with QuikChange Multi Site Directed Mutagenesis kit(Stratagene), according to manufacturer's instruction. The primers areas following: control mutant D177A, S178L, T180P (forward:5′-GGATGAAGAAATTTCCGGCATTAGAACCAAAAGAATGTCC, reverse:5′-GGACATTCTTTTGGTTCTAATGCCGGAAATTTCTTCATCC); E201K mutant (forward:5′-GATGAATCTTAAGGCCACTTTCTGCTTGGG, reverse:5′-CCCAAGCAGAAAGTGGCCTTAAGATTCATC); K268E, K270E mutant (forward:5′-GGCCAATGCCGAAGTCGAACTTTCCCTCCC; reverse:5′-GGGAGGGAAAGTTCGACTTCGGCATTGGCC). To develop the triple mutant (E201K,K268E, K270E), primers of mutant K268E, K270E and E201K were used as thetemplate. In order to verify their fidelity, the constructs weresequenced. The constructs were transformed into E. coli BL21 cells andexpressed and purified, according to manufacturer's instructions (GEHealthcare).

Adhesion Assay—

Assays utilizing endogenous ECM proteins generated by MCF10A cells wereperformed as previously described (Langhofer et al. (1993) J Cell Sci105 (Pt 3), 753-764; herein incorporated by reference in its entirety).MCF10A cells were plated in 96-well dishes and allowed to reachconfluence. Cells were washed with PBS and treated for 5 min with freshsterile 20 mM NH4OH, followed by extensive water washes. Wells wereblocked with heat-denatured BSA (10 mg/ml) for 1 hr at room temperature.Subconfluent cultures were trypsinized, washed with 37° C. serum-freeDMEM/F12 medium, and incubated with either antibodies or recombinantproteins (500 nM) for 30 min at 37° C. (when assaying endogenous maspinenzyme-free cell dissociation solution was used instead of trypsin). Inall assays, 2.0×104 cells were plated in triplicates and allowed toadhere for 30 min at 37° C. The GST protein was used as a control. Wellswere washed with 37° C. serum-free DMEM/F12 and adhered cells were fixedwith 5% gluteraldehyde and stained with crystal violet dye. Celladhesion was determined by the reading at 590 nm subtracted by the blankvalue (determined by BSA-coated wells, 5% of maximal cell adhesion).Cell adhesion was plotted as percentage of the corresponding controlvalue. Cell surface stripping of uPA from uPAR was conducted aspreviously described (Stoppelli et al. (1986) Cell 45, 675-684; hereinincorporated by reference in its entirety). Subconfluent cultures werewashed twice with DMEM/F12 supplemented with 20 mM HEPES and 1 mg/mlBSA. Then, cells were incubated with 50 mM glycine-HCl (pH 3.0) with 100mM NaCl at 25° C. for 2 min and the reaction was halted by neutralizingwith 500 mM HEPES (pH 7.4). In the function blocking studies, cells werefirst incubated with the function blocking antiuPAR or control rabbitIgG for 10 min, uPA and recombinant GST-maspin was added for another 20min.

Maspin Binding Assay—

Bacterial recombinant GST-maspin was labeled with ¹²⁵I as describedpreviously (Conlon, M. (2002) Preparation of ¹²⁵ I-Labeled Peptides andProteins with High Specific Activity using IODO-GEN, 2nd Edition Ed. TheProtein Protocols Handbook (Walker, J. M., Ed.), Humana Press Inc,Totowa, N.J.; herein incorporated by reference in its entirety). Wildtype (WT) and uPAR−/− mouse embryo fibroblasts (MEFs) (5.0×104 cells)were cultured in DMEM with 10% FBS on 96-well plate overnight. Cellswere chilled on ice for 30 min, washed three times with ice-cold DMEM,and then blocked on ice for 60 min using blocking buffer (DMEMcontaining 5% heat inactivated BSA). MEFs were incubated with increasingconcentrations of ¹²⁵I-GST-maspin in blocking buffer at 4° C. for 90min. After incubation, unbound ¹²⁵I-GST-maspin protein was removed bywashing three times with blocking buffer. Specific ¹²⁵I-GST-maspinbinding was determined by subtracting the detected radioactivity by thenonspecific binding (determined in the presence of a 50-fold excess ofnon-labeled GST-maspin). In the studies evaluating the uPA/uPARinteractions, ¹²⁵I-GST-maspin was treated with ABS4A and S-20 (SantaCruz) antibodies, which block the RCL and N-terminal domains, prior toadding to WT MEF cells. In one set, WT MEF cells were treated with ananti-mouse uPAR antibody (R&D), which blocks uPA binding to uPAR. In theother set, WT MEF cells were stripped of uPA, then treated with theantimouse uPAR antibody, and then administered uPA (800 nM) and¹²⁵I-GST-maspin. Results were reported as ¹²⁵I-GST-maspin binding to WTMEF cell surface as a percentage of control (IgG) antibody treatment.

Immunoprecipitation and Western Blot—

uPAR was overexpressed in MCF10A cells. Lysates from MCF10A cellsoverexpressing uPAR were prepared in modified radio-immunoprecipitationassay (RIPA) buffer: 50 mM Tris (pH 7.4), 1% Triton X-100, 1% sodiumdeoxycolate, 150 mM NaCl, 5 mM EDTA (pH 8.0), 5 mM PMSF, and proteaseinhibitor cocktail (Thermo Scientific). Cellular debris was cleared fromlysates by centrifugation and protein concentration was determined bythe BCA Protein Assay (Pierce). Whole cell extracts (500 μg) wereincubated overnight (constant rocking) with 5 μg of specific antisera orcontrol rabbit irrelevant antiserum (Ki67) at 4° C. ProteinA-Sepharose-coupled beads (Amersham Pharmacia Biotech) were added andincubated for 2 hr at 4° C. under constant agitation. Beads werecentrifuged, washed three times with ice-cold lysis buffer, and boiledfor 5 min in sample buffer containing 5% β-mercaptoethanol. Samplesseparated on SDS-PAGE gels, transferred to a PVDF membrane (GEHealthcare), and probed for β1 integrin, uPAR and maspin. Appropriatesecondary antibodies were added and proteins were visualized withenhanced chemiluminescence substrate (Pierce).

Example 2 Maspin is Secreted/Released from Cells

MCF10A cells were tested to determine if maspin is secreted/releasedinto culturing media. The culturing media was obtained from twodifferent MCF10A populations; i) cells obtained directly from ATCC orii) cells that have been cultured in the laboratory for two years, andanalyzed by Western immunoblot for maspin protein expression. Maspin wasdetected in all media and cells tested (SEE FIG. 1A). Using densitometryanalysis, it was determined that media from high passage MCF10A cellshad reduced maspin expression compared to the media from low passagecells. However, cellular maspin expression did not statistically differbetween low- and high-passage MCF10A. These results indicate that maspinis secreted/released from MCF10A cells and the loss of maspin secretioncould be an artifact of extended cell culturing conditions.

Example 3 Two Proximal Sites to the Reactive Center Loop are Responsiblefor Cell Adhesion

MCF10A cells deposit ECM proteins and can nucleate adhesive complexestypical of epithelia (Goldfinger et al. (1998) J Cell Biol 141, 255-265;Goldfinger et al. (1999) J Cell Sci 112 (Pt 16), 2615-2629; hereinincorporated by reference in their entireties). To determine theregion(s) of maspin that are involved in adhesion, competitive peptideswere used. First, peptides were developed that dissect theaforementioned region (amino acid residues 139-225) into peptidescorresponding to amino acid residues; 137-158, 169-189, 181-202,190-211, and 203-225. Then, using Molsoft ICM software to analyze thetertiary structure of maspin, peptides were developed to other regionsof maspin that are exposed such as the RCL (329-343), 260-275 and thecontrol peptide (97-112). Using these peptides, the regions of maspininvolved in mediating MCF10A cell adhesion were determined. The onlypeptides (derived from amino acid residues 139-225) that competed withGST-maspin were the 181-202 and 190-211 (SEQ ID NO:4;TDTKPVQMMNMEATFCMGNIDSI) peptides (SEE FIG. 2A). These results haveidentified the 190-202 region of maspin is partially responsible formediating cell adhesion. In addition, a novel region between amino acids260-275 was identified that is also important in regulatingmaspin-mediated MCF10A cell adhesion (SEE FIG. 1A). Although the 190-202and 260-275 peptides are separated by 58 amino acids in the primarystructure of maspin, they are adjacent to one another in the tertiarystructure (SEE FIG. 2B).

To further demonstrate that these regions are involved inmaspin-mediated adhesion, mutants of GST-maspin were developed; singlemutant (E201K), double mutant (K268E, K270E), triple mutant (E201K,K268E, K270E) and a control mutant (D177A, S178L, T180P). The single(E201K) mutant corresponds to peptide 181-202 while the double (K268E,K270E) mutant corresponds to peptide 260-275. The control (D177A, S178L,T180P) mutant failed to have any reduced cell adhesion. However, boththe single and double GST-maspin mutants had significantly reducedMCF10A cell adhesion (by 16% and 17%, respectively), as compared to WTGST-maspin (SEE FIG. 1C). The triple (E201K, K268E, K270E) mutant had anadditive effect whereby MCF10A cell adhesion was decreased byapproximately 28% when compared to WT GST-maspin (SEE FIG. 2C). Thesefindings demonstrate that proximal amino acid residues from 190-202 and260-275 in maspin mediate its effect on MCF10A cell adhesion.

Example 4 Maspin Localization to the Cell Surface Requires the uPA/uPARComplex

Recent accumulating evidence suggests an important role of the uPA/uPARcomplex in regulating cell adhesion (Smith & Marshall, C. J. (2010) NatRev Mol Cell Biol 11, 23-36; Dass et al. (2008) Cancer Treat Rev 34,122-136; herein incorporated by reference in their entireties). Maspinis localized with the uPA/uPAR complex at the cell surface (Yin et al.(2006) Cancer Res 66, 4173-4181; incorporated herein by reference in itsentirety); it was investigated whether uPAR is involved in maspinbinding to the cell surface. Using WT and uPAR−/− MEFs, it was foundthat WT MEFs displayed specific 125I-GST-maspin binding while uPAR−/−MEFs were absent of any specific 125I-GST-maspin binding, usingconcentrations within the normal physiological range (<500 nM) (SEE FIG.3A). These results indicate that uPAR expression is necessary for maspinto bind or localize to the cell surface.

The binding of ¹²⁵I-GST-maspin to WT MEF cells that were incubated witheither control (IgG), RCL (ABS4A) or N-terminal (S-20) blockingantibodies was examined Only the RCL (and/or surrounding area) and notthe N-terminus of maspin was found to be necessary for maspin binding tothe surface of WT MEFs (SEE FIG. 3B).

To determine if the uPA/uPAR complex is required for ¹²⁵I-GST-maspinbinding to MEF cell surface, an antibody that disrupts uPA binding touPAR was used. In the first set of experiments, treatment with thisantibody did not change the binding of ¹²⁵I-GST-maspin to WT MEF cells(SEE FIG. 3C, first set). However, ¹²⁵I-GSTmaspin binding to WT MEF cellsurface is reduced when cells were stripped of uPA and incubated withthe uPAR blocking antibody prior to re-introducing exogenous uPA and125IGST-maspin (SEE FIG. 3C). These results demonstrated that cellsurface localization of maspin requires not only uPAR but the uPA/uPARcomplex.

Example 5 Removal of Pericellular uPA Mitigates Maspin-Mediated CellAdhesion

Maspin binds to both uPA and pro-uPA causing reduced cell attachment bystrengthening mature focal adhesion contacts (Yin et al. (2006) CancerRes 66, 4173-4181; incorporated herein by reference in its entirety).Experiments conducted during development of embodiments of the presentinvention have demonstrated that the amino acid residues from 190-202and 260-275 in maspin mediate its effect on cell adhesion and theseregions are proximal to the RCL (SEE FIG. 2). To whether the associationof maspin and uPA may mediate cell adhesion, endogenous uPA wasacid-stripped prior to the addition of GST-maspin and evaluated theeffect on MCF10A cell adhesion. Stripping uPA substantially blocksmaspin-induced MCF10A cell adhesion (SEE FIG. 4A). In order to verifythat this ablation of maspin-induced adhesion was a direct effect of uPAremoval, uPA-stripped MCF10A cells were resupplied with exogenous uPA(800 nM). Addition of exogenous uPA rescues the maspin-mediated adhesionof uPA-stripped MCF10A cells (154.06% of GST control, SEE FIG. 4A).These experiments demonstrate that uPA is needed for maspin-mediatedadhesion.

Example 6 The uPA/uPAR Complex is Necessary for Maspin-Mediated CellAdhesion

These combined studies revealed that uPAR or uPA removal from thesurface of MCF10A cells ablates maspin localization to the cell surface(SEE FIG. 3) and its ability to enhance cell adhesion (SEE FIG. 4A).Experiments were conducted during development of embodiments of thepresent invention to determine whether uPA and uPAR functionindependently or if the localization and adhesion functions of maspinrequire the uPA/uPAR complex. uPAR blocking antibody (antiuPAR, blocksfree uPA from binding to uPAR) was used to elucidate the role(s) of theuPA/uPAR complex in maspin-mediated adhesion. Endogenous uPA wasstripped from the cell surface and then the cells were incubated withcontrol rabbit IgG or anti-uPAR for 10 min, and finally after theaddition of exogenous uPA (800 nM) and GST-maspin (500 nM) the cellswere allowed to adhere. At a lower concentration of the anti-uPAR (0.25mg/ml), there was a slight decrease in cell adhesion, albeit notsignificant. However, at higher concentration of anti-uPAR (0.5 and 1.0mg/ml), maspin-mediated MCF10A cell adhesion was significantly inhibitedcompared to control antibody treatment (SEE FIG. 4B). These findingsdemonstrate that disrupting uPA binding to uPAR impairs maspin-mediatedadhesion. If MCF10A cells are not stripped of uPA, addition of theblocking uPAR antibody has no effect on maspin-mediated cell adhesion(SEE FIG. 4C). The inability of the uPAR blocking antibody to reduceMCF10A cell adhesion implicates the uPA/uPAR complex as effectors ofmaspin-mediated cell adhesion.

Example 7 Maspin Forms a Complex with uPAR and β1 Integrin

Experiments were conducted during development of embodiments of thepresent invention to detect physical association of maspin with bothuPAR and 131 integrin to produce a complex that regulates both cellmotility and adhesion. The biophysical interaction between maspin, uPAR,and β1 integrin was detected by immunoprecipitating cell lysates fromMCF10A cells overexpressing uPAR (SEE FIG. 5). Maspin co-precipitatedwith both uPAR and β1 integrin. These are the first studies showing theassociation of all three proteins, the maspin-uPAR-β1 integrin complex.

Example 8 Anti-Angiogenesis Mapsin Peptides

Experiments were conducted during development of embodiments of thepresent invention to develop small maspin mimetic peptides fortherapeutic treatment. Through maspin deletion and site-specificmutagenesis analysis, the unique region/sites within maspin protein havebeen identified that are responsible for its binding to cell surface andthe mechanism of maspin-mediated signal transduction (SEE FIG. 6). Basedon this information, two competitive peptides were designed that act asthe antagonist that blocks wildtype maspin's effect on cell adhesion inthe range of 0.10-1.0 uM. These two peptides, containing 15 and 22 aminoacids respectively, are located in two different regions in primaryprotein sequence but are spatially very close to each other in thefolded protein. Synthetic peptides were tested that contain the keyamino acids in both the parental peptides. Amino acid residues weremutated in certain sites in the middle of the peptides. To make thepeptides more stable, small peptides were designed (e.g., smaller than12 amino acids) and were capped at both ends with protective moieties.Peptides were synthesized in high purity and tested in biologicalassays.

HUVEC endothelial cells were used to test the effect of synthetic maspinpeptides on HUVEC tube formation assay in cell culture systems. Randompeptide was used as a negative control and maspin G-helix peptide as apositive control. G-Helix was previously found to be effective ininhibiting endothelial cell migration, thereby mimicking maspin effectagainst angiogenesis. G-Helix had a modest effect on HUVEC tubeformation at the concentration of 10 uM. At 1.0 uM, G-helix was noteffective. However, two of the newly identified peptides: peptide X(STANAKVKLSIP (SEQ ID NO:5)) and peptide Y (TANAEVKLSIPK (SEQ ID NO:6)are highly effective in inhibiting tube formation at a low concentrationof 1.0 uM (SEE FIG. 7).

Based on the experiments conducted during development of embodiments ofthe present invention, it is further contemplated the peptide ZSTENAKVKLSIP (Seq ID NO. 7) would also be effective in enhancing celladhesion and/or inhibits cell motility; inhibiting tube formation;associating with uPAR, 131 integrin, or both; localizing at the cellsurface; co-localizing with the uPA/uPAR complex; and/or treatingcancer, metastasis, and/or bone formation disorders.

REFERENCES

All publications and patents mentioned in the present application and/orlisted below are herein incorporated by reference in their entireties.

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What is claimed is:
 1. A composition comprising a peptide or polypeptidethat comprises at least 70% sequence identity with all or a portion ofone or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, and SEQ ID NO:7.
 2. The composition of claim 1,wherein said peptide or polypeptide is at least 6 amino acids in length.3. The composition of claim 2, wherein said peptide or polypeptide is8-20 amino acids in length.
 4. The composition of claim 2, wherein saidpeptide or polypeptide comprises at least 90% sequence identity with allor a portion of one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.
 5. Thecomposition of claim 4, wherein said peptide or polypeptide comprises atleast 90% sequence identity with SEQ ID NO:5.
 6. The composition ofclaim 4, wherein said peptide or polypeptide comprises at least 90%sequence identity with SEQ ID NO:6.
 7. The composition of claim 4,wherein said peptide or polypeptide comprises at least 90% sequenceidentity with SEQ ID NO:7.
 8. A pharmaceutical composition comprising:(a) one or more of: ((i) a peptide or polypeptide with at least 70%sequence identity with all or a portion of SEQ ID NO:1, (ii) a peptideor polypeptide with at least 70% sequence identity with all or a portionof SEQ ID NO:2, (iii) a peptide or polypeptide with at least 70%sequence identity with all or a portion of SEQ ID NO:3, (iv) a peptideor polypeptide with at least 70% sequence identity with all or a portionof SEQ ID NO:4, (v) a peptide or polypeptide with at least 70% sequenceidentity with all or a portion of SEQ ID NO:5, (vi) a peptide orpolypeptide with at least 70% sequence identity with all or a portion ofSEQ ID NO:6; (vii) a peptide or polypeptide with at least 70% sequenceidentity with all or a portion of SEQ ID NO:7; and (b) a physiologicallysuitable buffer.
 9. A method of enhancing cell adhesion comprisingadministering a composition of claim 1 to a cell, tissue, or subject.10. The method of claim 9, wherein enhancing cell adhesion results inreduced cell motility.
 11. The method of claim 9, wherein said cells,tissue, or subject comprises cancer cells, cells at risk of becomingcancerous, or cells at risk of metastasis.
 12. The method of claim 9,wherein said peptide or polypeptide physically associates with uPAR, β1integrin, or both upon administering to said cell, tissue, or subject.13. The method of claim 9, wherein said peptide or polypeptide localizesat the cell surface upon administration to said cells, tissue, or asubject.
 14. The method of claim 9, wherein said peptide or polypeptideco-localizes with the uPA/uPAR complex.
 15. A method of treating orpreventing cancer or metastasis comprising administering to a cell,tissue, or subject a composition of claim 1, wherein administering saidpeptide or polypeptide enhances cell adhesion and/or inhibits cellmotility.
 16. A method of treating or preventing a bone formationdisorder comprising administering to a cell, tissue, or subject acomposition of claim 1.